This project includes the development of assays to phenocopy inherited genetic mutations leveraging disease knowledge from basic and clinical research programs with recent advances in molecular biology (e.g., application of TALEN and CRISPR-mediated genome editing). The assay designs are considered in the context of analysis and progression strategies for evaluation of approved drugs and investigational agents using HTS technologies. The lab has a strong emphasis on methods development research to advance assay and screening efficiency in drug discovery and chemical genomics. Peroxisome biogenesis disorders (PBDs): In collaboration with J. Hacia (USC), N. Braverman (U. Toronto) and S. Steinberg (JHU), the ADST laboratory of NCATS is optimizing a 1,536-well plate format high-content imaging assay for testing across chemical libraries at NCATS, including a repository of approved drugs and collections of diverse chemical substances. Peroxisome biogenesis disorders (PBDs) are autosomal recessive disorders (overall incidence of 1;50,000 U.S. births) caused by defects in PEX genes encoding proteins required for normal peroxisome assembly. Severely affected patients show profound mental retardation secondary to neuronal migration defects and hypomyelination, and die by one year of age; however, the majority of patients have milder forms of disease and survive through adulthood. These individuals show mild to moderate mental retardation, craniofacial dysmorphism, liver dysfunction, progressive sensorineural hearing loss and retinopathy. Currently, disease management is only supportive in nature. The diagnosis of relatively asymptomatic children presents a challenge to identify treatments that can halt disease progression, which will become more urgent with the emergence of newborn screening programs. AMP deaminase (AMPD) deficiency: In collaboration with K. Nagaraju (Children's National Medical Center), the ADST laboratory is developing a coincidence reporter assay to aid in the discovery of compounds that increase expression of the enzyme AMPD implicated in the disease idiopathic inflammatory myositis (IIM). IIM is a rare autoimmune progressive disease that afflicts the skeletal muscle of patients. IIM is comprised of errors in both immune regulation and intrinsic muscle metabolism. AMPD catalyzes the deamination of AMP to IMP. In humans, AMPD activity is encoded by at least three genes. AMPD isoforms have tissue specific expression patterns in adults and stage specific expression patterns during the muscle development. Drugs which stimulate the expression of AMPD1 are hypothesized to reverse muscle weakness. Diamond Blackfan Anemia (DBA): In collaboration with D. Bodine (NHGRI, NIH) and G. Thomas (U. Cincinnati), the ADST laboratory is developing assay strategies as part of a translational research project for DBA a rare, congenital disease seen in all ethnic groups with a frequency of 7 cases per million live births. The diagnosis is usually made in the first year of life, as a severe anemia that requires transfusion. Unlike other anemias, DBA patients have no reticulocytes in peripheral blood indicating a failure of erythropoiesis, whereas production of the white cells and platelets are unaffected. For a small minority of patients, bone marrow transplantation from a healthy sibling is a curative therapy. For patients without a suitable donor, a variety of palliative therapies that can prolong life into the third decade. Current treatments include corticosteroid therapy, which results in reduced growth and other complications, or lifelong transfusion and iron chelation. Discovery of Drugs for inherited rare blinding retinal degenerations: Donald J. Zack, M.D., Ph.D., a Johns Hopkins researcher funded by the Foundation Fighting Blindness, is collaborating with Dr. Ingleses ADST laboratory in a public-private partnership aimed at alleviating and curing blinding retinal degenerative diseases. Together, they are utilizing ADST researchers expertise in assay development, chemical biology and quantitative high-throughput screening (qHTS) to develop assays and chemical library testing paradigms to identify drugs and compounds that have the potential to save and restore vision for people affected by devastating retinal diseases such as retinitis pigmentosa and age-related macular degeneration, the leading cause of blindness in Americans over 50 years of age. The use of primary rodent retinal cells, a limiting cell type typical for typical HTS experiments, benefits from the low-volume assay technology employed in the Inglese laboratory at NCATS. Development of a cell-based high-throughput quantitative microscopy-based assay for glucose-regulating multienzyme compartment: In collaboration with S. An (UMBC), the ADST laboratory of NCATS is developing a 1,536-well plate format high-throughput quantitative microscopy-based assay for discovery of compounds that modulate the formation of a dynamically assembled multienzyme complex regulating glucose-derived carbon flux in cells. Given that glucose metabolism is the central metabolic pathway that balances the cellular needs for both energy and building blocks, the pilot screening of pharmacologically active small molecules for or against the assembly will assist to understand the biological significance of the assembly in the cell. In turn, extensive HTS is anticipated to discover novel pharmacophores, which promote or disrupt the assembly in human disease models, for therapeutic intervention in the treatment of glucose metabolism-associated human diseases. Exploration of novel sources of chemical libraries for antimalarial activity: To probe the biological activity of the stereochemical and structural complexity of novel academic chemical libraries and the chemical diversity of natural products extracts (NPEs) the ADST laboratory is testing the antimalarial potential of these chemical repositories. In a multi-investigator collaboration effort with D. Sherman (U. Michigan), J. Clardy (HMS), J. Porco (BU), S. Schaus (BU), A. Beeler (BU), J.K. Snyder (BU),and X.-z., Su (NIAID, NIH), the ADST laboratory has determined EC50s for Plasmodium falciparum viability for each of 2,070 members of the Boston University CMLD compound collection and >16,000 NPEs assembled at the University of Michigan using qHTS across five parasite lines of distinct geographic origin. From the BU library, three compound classes displaying either differential or comprehensive antimalarial activity across the lines were identified. From the NPE library a novel recently discovered compound, called Actinoramide A was found to display potent antimalarial activity. The molecular basis for the activity of these compounds is being investigated.